title: Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis.
abstract: GRASP65 (Golgi reassembly and stacking protein of 65 KDa) is a cis-Golgi protein with roles in Golgi structure, membrane trafficking and cell signalling. It is cleaved by caspase-3 early in apoptosis, promoting Golgi fragmentation. We now show that cleavage is needed for Fas-mediated apoptosis: expression of caspase-resistant GRASP65 protects cells, whereas expression of membrane proximal caspase-cleaved GRASP65 fragments dramatically sensitises cells. GRASP65 coordinates passage through the Golgi apparatus of proteins containing C-terminal hydrophobic motifs, via its tandem PDZ type 'GRASP' domains. Fas/CD95 contains a C-terminal leucine-valine pairing so its trafficking might be coordinated by GRASP65. Mutagenesis of the Fas/CD95 LV motif reduces the number of cells with Golgi-associated Fas/CD95, and generates a receptor that is more effective at inducing apoptosis; however, siRNA-mediated silencing or expression of mutant GRASP65 constructs do not alter the steady state distribution of Fas/CD95. We also find no evidence for a GRASP65-Fas/CD95 interaction at the molecular level. Instead, we find that the C-terminal fragments of GRASP65 produced following caspase cleavage are targeted to mitochondria, and ectopic expression of these sensitises HeLa cells to Fas ligand. Our data suggest that GRASP65 cleavage promotes Fas/CD95-mediated apoptosis via release of C-terminal fragments that act at the mitochondria, and we identify Bcl-X(L) as a candidate apoptotic binding partner for GRASP65.
authors.affiliation: Cell Biology Laboratories, School of Biochemistry, University of Bristol, UK.
authors.forename: J P X
authors.initials: JP
authors.lastname: Cheng
authors.forename: V M S
authors.initials: VM
authors.lastname: Betin
authors.forename: H
authors.initials: H
authors.lastname: Weir
authors.forename: G M A
authors.initials: GM
authors.lastname: Shelmani
authors.forename: D K
authors.initials: DK
authors.lastname: Moss
authors.forename: J D
authors.initials: JD
authors.lastname: Lane
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology; D051020:bcl-X Protein / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D053222:Fas Ligand Protein; C108705:GORASP1 protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D051020:bcl-X Protein; D019014:fas Receptor; D019284:Thapsigargin; D020169:Caspases